The influence of cryopreservation on a benzimidazole-resistant isolate of Haemonchus contortus conditioned for inhibited development

Larvae of a fenbendazole (FBZ)-selected, benzimidazole-resistant strain of Haemonchus contortus were conditioned for inhibited development during 7 weeks at 15‡ C. Sheep raised nearly wormfree were infected with nonfrozen larvae (L3). Larvae from the same batch were exsheathed and cryopreserved over liquid nitrogen for 1 year, and another group of sheep was infected with the same dose of these L3 larvae after thawing. Egg hatch assays for all sheep were done between 21 and 30 days post infection (p.i.). Fenbendazole susceptibility was tested by treating 50% of each group of sheep on day 30 p.i. Worm counts of all sheep were done on day 38 p.i. The exsheathment time of the designated frozen L3 larvae at 15‡ C exceeded the usual time span at room temperature. Of the frozen L3 larvae, 60% was motile after thawing. The mean LC₅₀ values (?g tiabendazole/ml) and the mean percentage of efficacy after FBZ treatment against total worm burden, adults and early L4 larvae of nonfrozen and frozen H. contortus were not significantly different. The mean percentages of take (infectivity) for nonfrozen and frozen doses were 35.3% and 5.9%, respectively (P}<0.001). In nontreated sheep, the mean percentages of inhibition for nonfrozen and frozen doses were 91.9%, and 54.2%, respectively (P<0.01). The exsheathment time seems to be temperature-dependent. The induction of inhibited development of L3 larvae during 7 weeks was as effective as during 5 weeks at 15‡ C. Cryopreservation did not affect the susceptibility for benzimidazoles in vivo or in vitro; for this reason, it is a reliable method for storing drug-resistant strains of H. contortus. Impairment of the infectivity of this prolific species was not dramatic. This first report on the influence of cryopreservation on inhibition-proneness shows that deep-freeze storage might influence the level of this characteristic; however, other factors might be involved.